Reclassification of eight Akkermansia muciniphila strains and description of Akkermansia massiliensis sp. nov. and Candidatus Akkermansia timonensis, isolated from human feces

Akkermansia muciniphila is a human intestinal tract bacterium that plays an important role in the mucus layer renewal. Several studies have demonstrated that it is a modulator for gut homeostasis and a probiotic for human health. The Akkermansia genus contains two species with standing in nomenclature but their genomic diversity remains unclear. In this study, eight new Akkermansia sp. strains were isolated from the human gut. Using the digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI) and core genome-based phylogenetic analysis applied to 104 A. muciniphila whole genomes sequences, strains were reclassified into three clusters. Cluster I groups A. muciniphila strains (including strain ATCC BAA-835T as type strain), whereas clusters II and III represent two new species. A member of cluster II, strain Marseille-P6666 differed from A. muciniphila strain ATCC BAA-835T and from A. glycaniphila strain PytT in its ability to grow in microaerophilic atmosphere up to 42 °C, to assimilate various carbon sources and to produce acids from a several compounds. The major fatty acids of strain Marseille-P6666 were 12-methyl-tetradecanoic and pentadecanoic acids. The DNA G + C content of strain Marseille-P6666 was 57.8%. On the basis of these properties, we propose the name A. massiliensis sp. nov. for members of cluster II, with strain Marseille-P6666T (= CSUR P6666 = CECT 30548) as type strain. We also propose the name “Candidatus Akkermansia timonensis” sp. nov. for the members of cluster III, which contains only uncultivated strains, strain Akk0196 being the type strain.


Results and discussion
Strain identification and phylogenetic analyses. All strains isolated in this study were first identified as Akkermansia muciniphila by MALDI-TOF-MS when we comparing their peptidic profiles to those available in the Bruker database. After sequencing the 8 strains, the 16S rRNA sequences of each isolate were extracted and compared to those of closely related species in the NCBI database (https:// www. ncbi. nlm. nih. gov/). Phenotypic and biochemical analysis. Cells from strain Marseille-P6666 were rod-shaped (0.5 × 0.8 μm), motile and Gram-negative (Fig. 1). In the presence of fluid, the cells turn on themselves and self-propel. Multiple cilia can be observed on the bacterial cell surface (Fig. 1). Colonies grown on Columbia agar plates appeared white, non-haemolytic, and circular with a diameter of 0.5 mm after 72 h of incubation. Optimal growth from strain Marseille-P6666 grew between from 37 to 42 °C, at a pH ranging from 6 to 7.5, and in the presence of 0 to 5 g/l NaCl. Strain Marseille-P6666 was able to grow in microaerophilic atmosphere, which enables it to survive in the mucus layer of the gastrointestinal tract 23 . The new isolate was able to use mucin as a solo carbon source.
Cells were catalase-positive and oxidase-negative. Using an API ZYM strip (bioMérieux), production of alkaline phosphatase, esterase (C4), acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase and N-acetyl-β-glucosaminidase were positive. Using an API 20NE strips (bio-Mérieux), strain Marseille-P6666 was able to hydrolyze esculin and to produce β-galactosidase. Using an API 20A strip (bioMérieux), positive reactions were obtained for acidification of d-glucose, d-mannitol, d-lactose, d-maltose, esculin ferric citrate and d-mannose. Using an API 50CH strips (bioMérieux), strain Marseille-P6666 utilized l-arabinose, d-ribose, d-galactose, d-glucose, d-fructose, d-mannose, d-mannitol, d-sorbitol, N-acetylglucosamine, salicin, esculin ferric citrate, cellobiose, d-maltose, d-lactose, d-melibiose, amygdaline, d-saccharose, d-trehalose, arbutine, gentiobiose, d-turanose, d-tagalose and potassium gluconate as sole carbon sources. All negative properties obtained from the API ZYM, 50CH, 20A and 20NE strips were summarized in the description of the novel species. Furthermore, the physiological and biochemical characteristics of strain Marseille-P6666 T were summarized and compared to those of other closely related species in Table 1. Strain Marseille-P6666 was found to be susceptible to trimethoprim-sulfamethoxazole, doxycycline, rifampicin, clindamycin, amoxicillin, oxacillin and benzylpenicillin but susceptible to vancomycin, amikacin, ciprofloxacin, tobramycin, ceftriaxone and ceftazidime. The major cellular fatty acids of strain Marseille-P6666 were saturated structures: 12-methyl-tetradecanoic acid (58.4%), pentadecanoic acid (15.8%) and 12-methyl-Tridecanoic acid (6%). The two major fatty acid namely anteiso-C 15:0 and C 15:0 are similar for strains Marseille-P6666, Muc T and Pyt T (Table 2). Short fatty acids such as acetic acid (12 ± 7 mM) and propanoic acid (5 ± 2 mM) were produced. Strain Marseille-P6666 produced various polar lipids classes such as sphingomyelins, N-acyl ethanolamines, acyl carnitine, phosphatidylethanolamine, Lysophosphatidyléthanolamine, phosphatidylcholine, lysophosphatidylcholine, ceramides-glycero lipids, fatty acyls-glycero lipids, phosphatidic acid and several unknown structures and phospholipids (Supplementary Fig. 1 Table 3. Genome sequences of these isolates had different sizes ranging from 2,740,501 to 3,280,190 bp. The genome of Marseille-P6666 is 3,280,190 bp long with an average G + C content of 57.8%. It is composed of three contigs. Of the 2793 predicted genes, 2726 were protein-coding genes, 9 RNAs, 1 tmRNA and 57 tRNA genes. Circular maps of this strain are illustrated in Fig. 2. The genome of Marseille-P6666 is greater than that of A. muciniphila ATCC BAA-835 T (= Muc T ), (2,664,051 bp) and A. glycaniphila pyt T (3,074,078 bp). The G + C content of Marseille-P6666 is larger than that of A. muciniphila ATCC BAA-835 T (55.6%) but smaller than that of A. glycaniphila Pyt T (58.2%). Distribution of genes into COGs functional categories between Marseille-P6666 and the other closely related species was presented in Fig. 3 and Table 4. The number of genes from each COG category was greater for strain Marseille-P6666 than for A. muciniphila strain ATCC BAA-835 T , notably for genes encoding cell wall and membrane biogenesis, energy production and conversion, defense mechanisms, and transport and metabolism of carbohydrates, amino acids, nucleotides and inorganic ions. This is consistent with the fact that strain Marseille-P6666 has more coding genes (1983 genes) than A. muciniphila strain ATCC BAA-835 T (1084 genes). The phylogenetic tree based on core genome identified tree clusters (I, II, and III), with strains considered previously belonging to the A. muciniphila species. Among the Akkermansia strains isolated in this study, six of them, clustered with A. muciniphila strain ATCC BAA-835 T (Cluster I). Two strains formed a second cluster (Cluster II) with other strains previously classified as belonging to the A. muciniphila species. Comparison of the genomes from members of the clusters I, II and III with A. muciniphila strain ATCC BAA-835 T , showed dDDH values higher than 70% with cluster I members (range 74.80% to 100%) but lower than 70% for cluster II and III members (range 33.8-34.2% and 17.1-24.9%, respectively, Supplementary Table 2). ANI values between cluster I, II and III isolates were 97-100%, 88% and 82% with A. muciniphila ATCC BAA-835 T , respectively ( Supplementary Fig. 2). ANI values between the three clusters (I, II and III) were significantly lower than the proposed cutoff value of 95% for defining a bacterial species 24,25 . A recent study even redefined the ANI threshold value to 96.5% for creating a new bacterial species 26 . In contrast, all ANI values within a given cluster were higher than 97%. Therefore, the distribution of strains previously considered as A. muciniphila into three distinct species was clearly supported by the genome-based phylogenetic analysis and the of DDH and ANI values.
Hence, our genome analysis results strongly suggest that strains currently classified as A. muciniphila belong to three distinct species. However, the 98.7% 16SrRNA sequence similarity threshold defined to classify a bacterial species 27 cannot discriminate between species in the Akkermansia genus. This highlights the limitation of 16S rRNA gene analysis for the correct species classification within some bacterial genera ( Supplementary  Fig. 3) 28 . Cluster I is formed by A. muciniphila strains, including the type strain ATCC BAA-835 T (Fig. 4). Cluster II includes strains Marseille-P6666, Marseille-P9185 and 12 other strains described in previous studies (Fig. 4). Recently, Kumar et al. 29 described a new Akkermansia strain, DSM 33459, that was phylogenetically close to strains EB-AMDK-39, EB-AMDK-40, and EB-AMDK-41, and proposed that this strain belongs to a new Akkermansia species. According to these authors, genomic comparison showed that strain Akkermansia sp. DSM , and therefore could not be included in our analysis. In addition, the authors deposited strain DSM 33459 in the DSMZ collection, but not in a second culture collection as requested by rule 30 from the international code of nomenclature of Prokaryotes for the description of a new species 30 . In addition, no name was proposed by the authors for this new species and Kumar and colleagues' article does not contain any protolog to officially describe the properties of the new species. Cluster III includes the five strains Akk0196 T , Akk0490, Akk0496a, Akk0496b and Akk2750 (Fig. 4). We observed that A. glycaniphila strain Pyt T , initially described by Janneke et al., exhibited dDDH and ANI values of 22.5% and 73%, respectively, with A. muciniphila strain ATCC BAA-835 T19 . In addition, A. glycaniphila strain Pyt T also exhibited dDDH and ANI values ranging from 18.5 to 24% and from 73 to 74%, respectively, with all other members of the Akkermansia genus.
Pan-genome analysis of Akkermansia muciniphila, Akkermansia massiliensis sp. nov. and Candidatus Akkermansia timonensis sp. nov.. The pan-and core-genomes of A. muciniphila strains (85 strains) were composed of 6357 and 1193 genes, respectively. In addition, 1654 genes are accessory genes. A total of 1108 specific genes were found in only one A. muciniphila strain.
The pan-and core-genomes of A. massiliensis strains (14 strains) were composed of 3632 and 2138 genes, respectively. The accessory genome included 410 genes were strain-specific.   www.nature.com/scientificreports/ The pan-and core-genomes of A. timonensis strains (5 strains) were composed of 2767 and 2539 genes, respectively. The accessory genome sizes were 228 genes. A total of 125 specific genes were found in only one strain of A. timonensis.
The pan-genome of A. muciniphila ATCC BAA-835 T (6357genes) is larger than that of A. massiliensis Marseille-P6666 (3632 genes). The percentage of the core-genome of A. muciniphila ATCC BAA-835 T is smaller than that of A. massiliensis Marseille-P6666, 18.7% and 60.1%, respectively. However, due to the difference in the number of genomes used in the analysis of each pan-genome, the number of accessory genes of A. muciniphila ATCC BAA-835 T (85 strains) is higher than that of A. massiliensis Marseille-P6666 (14 strains), 26% and 11.2% of the pan-genome, respectively.

Conclusion
From these results, we suggested the creation of two new species: Akkermansia massiliensis sp. nov. that includes strains Marseille-P6666 and Marseille-P9185, for which Marseille-P6666 T is the type strain; and Candidatus Akkermansia timonensis sp. nov. that includes strains Akk0196, Akk0490, Akk0496a, Akk0496b and Akk2633.   www.nature.com/scientificreports/ Gram strain-negative, rod-shaped cells (0.5 × 0.8 μm). Bacteria are catalase-positive, oxidase-negative and motile. Non-spore forming. Colonies grown on Columbia agar are white, circular, convex and with entire margins and uniform. The optimal growth is observed in anaerobic atmosphere, at 37 °C, at pH 7 and in the presence of 5 g/l NaCl. Growth may also be obtained in microaerophilc atmosphere and at temperatures up to 42 °C. Nitrate reduction, indole production, gelatin hydrolysis and urease activities are absent. Strain Marseille-P6666 T is positive for esculin hydrolysis and exhibits α-galactosidase, β-galactosidase, β-glucuronidase,  Member of the Akkermansia genus, Candidatus Akkermansia timonensis is a bacterial species identified by metagenomic analyses. The genome length of the type genome is 3,212,887 bp and the G + C content is 56.7%.

Materials and methods
Isolation and identification of strains by MALDI-TOF. Stools obtained from eight French patients as part of a culturomics project aiming at isolating as many distinct human-associated bacterial species from the gut, were included in the study from 2017 to 2020. All the methods used in this study were carried out in accordance with relevant guidelines and regulations conformed to the Declaration of Helsinki. Informed and oral consent was obtained from the stool donors. Approximately 1 g of each feces specimen was suspended in 2 ml of phosphate-buffered-saline (Life, Technologies, Carisbad, CA, USA). Then, 100 µl of each stool suspension was tenfold diluted up to 10 -10 . After that, 50 µl was inoculated on 5% sheep blood-enriched Columbia agar (BioMérieux, Marcy l'Etoile, France) and incubated at 37 °C in anaerobic atmosphere generated by AnaeroGen generator (bioMérieux). After 72 h of incubation, single colonies were selected and subcultured on the same www.nature.com/scientificreports/ medium in order to obtain pure isolates. Strains were identified using a Microflex MALDI-TOF MS spectrometer (Bruker, Daltonics, Leipzig, Germany) as previously described 31 . Phenotypic and biochemical characterization. Cell morphology and characteristics of these isolates were observed using a TM4000 scanning electron microscope (Hitachi, Tokyo, Japan) from fresh colonies as previously described 30 . Colony morphology was described after observation of the strain grown after four days at 37 °C. Gram staining and spore formation were investigated and mobility was examined by microscopic observation 32 . Growth on Columbia agar at different temperatures (21 °C, 28 °C, 37 °C, 42 °C and 45 °C) and in microaerophilic conditions was tested using CampyGenTM (BioMérieux, ThermoFisher scientific) after 72 h of incubation. Growth in various NaCl concentrations (0, 5, 10 and 15 g/L) and at a pH range of 5 to 8.5 (at intervals of 0.5 pH unit) were assessed using Columbia agar plates 33 .
The ability to use mucin as sole carbon source was tested by using a modified basal media described by Derrien et al. 2  Catalase and oxidase activities were assessed by using a BBL™ DrySlide™ (Becton, Le Pont de Claix, France) according to the manufacturer's instructions. Activities of other enzymes and metabolic characteristics were investigated by using the API ZYM, API 50CH, API 20A and API NE strips according to the manufacturer's instructions (bioMérieux). Susceptibility to antibiotics was tested using the following E-test strip gradients: amoxicillin, benzylpenicillin, oxacillin, cefotaxine, ceftriaxone, amikacin, tobramicin, ciprofloxacin, clindamycin, doxycycline, rifampicin, vancomycin and trimethoprim-sulfamethoxazole. Plates with deposited strips were incubated at 37 °C for 48 h. Minimal inhibitory concentration (MIC) of each tested antibiotic was determined according to the manufacturer's instructions 34 . Chemotaxonomic characteristics. Cellular fatty acid methyl ester (FAME) analysis was performed by Gas Chromatography/ Mass Spectrometry (GC/MS) as previously described 35 . Approximately 65 mg of bacterial biomass collected from several Columbia agar plates cultured under anaerobic conditions for 3 days at 37 °C were distributed into each of two sterile tubes. FAMEs were extracted and prepared as described before by Sasser 36 . GC/MS analyses were done as previously described 35 .
Short chain fatty acids (SCFA) were extracted and analyzed from three independent culture bottles (both blank and samples). Strain Marseille-P6666 was cultured in anaerobic blood culture vial enriched with 5% sterilized sheep blood (Becton-Dickinson, Pont de Claix, France) for three days. SCFAs were measured with a Clarus 500 chromatography system connected to a SQ8s mass spectrometer (Perkin Elmer) as previously described by Diop et al. 37 .
Polar lipid analysis of strain Marseille-P6666 was performed by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry (HILIC-MS). Total lipids were extracted from cultures plates according to the Bligh and Dyer protocol 38 . Fifty percent chloroform/methanol was used to reconstitute the chloroformed extracts previously dried under a nitrogen stream, corresponding approximately to 0.5 mg of lipid content per 100 µL (v:v). Lipid extracts were injected (5 µL) into a HILIC column (BEH HILIC, 2.1 × 100 mm, 1.7 µm, Waters, Guyancourt, France). Elution of lipids from the column was performed according to their polarity using a gradient of the following solvent compositions: A = 5% water/95% acetonitrile, B = 50% H 2 O/50% acetonitrile, both at 10 mM ammonium acetate pH8. The HD-MS method (Vion ESI-IMS-Q-TOF mass spectrometer, Waters) with positive and negative modes was used for lipid control as previously described 39 . The assignment of lipid classes was done according to the retention times (RT) of an injected standard (Splash Lipidomix, Avanti Polar Lipids, Alabaster, AL, USA). A comparison of the corresponding masses with the COMP DB LipidMAPS database (tolerance of 0.0005 m/z; all chains are activated) was also performed to confirm the lipid classes.
Genome sequencing and assembly. Genomic DNAs (gDNAs) from all strains were extracted using an EZ1 biorobot and the EZ1 DNA Tissue kit (Qiagen, Hilden, Germany). The gDNAs were quantified by a Qubit assay with the high sensitivity kit (Thermofisher Scientific) to 0.2 ng/μl. gDNAs were sequenced using a MiSeq sequencer (Illumina, San Diego CA, USA) with the paired-end strategy. SPAdes was used to assemble the total reads of all genomes. Scaffolds smaller than 800 bp and those with depth values lower than 25% of the average depth (considered as possible contaminants) were deleted.
Genome annotation and comparison. Genome annotation was performed using the Prokka software 40 .
Bacterial protein-coding sequences were predicted using BLASTP (E-value of 1e-03, coverage 0.7 and identity 30%) against the Clusters of Orthologous Groups (COG) database. Graphical circular maps of genomes was generated using CGView (Circular Genome Viewer) software 41 .
As of February 22, 2022, 191 complete A. muciniphila genomic sequences were available in the NCBI Gen-Bank database and were downloaded. For genomic comparison, we eliminated duplicate sequences, retaining only 96 complete sequences from several studies (Supplementary Table 3). Overall, a total of 114 sequences were analyzed, including eight from this study, three from other Akkermansia species (A. glycaniphila, "Candidatus A. intestinigallinarum" and "Candidatus A. intestinavium"), and seven from closely related species from the Verrucomicrobiaceae family. Several genomic comparison approaches were used to delineate the species within the Akkermansia genus. The Genome-to Genome Distance Calculator (https:// ggdc. dsmz. de/) and PyANI (a Python package and script that provides support for calculating ANI) 42  www.nature.com/scientificreports/ hybridization (dDDH) and average nucleotide identity (ANI) between studied strains, retrospectively. The pangenome of Akkermansia strains was analyzed using the Roary software 43